Gastroenterology and Hepatology from Bed to Bench. ©2016 Rigld, Research Institute for Gastroenterology and Liver Diseases Helicobacter Pylori Caga Induced Interleukin-8 Secretion in Gastric Epithelial Cells Patients and Methods

Introduction Helicobacter pylori is colonized in the stomach of 35%-70% people worldwide. It is a Gram-negative bacterium that lead to disorders ranging from gastric inflammation to gastric adenocarcinoma (1-4). Several virulence factors of H. pylori are involved in the pathogenesis of the stomach. H. pylori chronic infection could increase expression of specific immune mediators, such as interleukins, tumor necrosis factor (alpha), and interferons in the host cells (5-8). Interleukin 8 (IL-8), a potent neutrophil chemotactic factor, plays a crucial role in the proliferation and migration of cancer cells in the infected patients. It also is able to stimulate the host specific inflammatory response through chemo attraction of neutrophils to the site of infected gastric mucosa and their activation (9, 10). In vivo and in vitro studies showed higher IL-8 secretion in the H. pylori infected cells and mainly correlated this elevated IL-8 level with expression of functional CagA, a major virulence factor of H. pylori which is located at the end of the cag pathogenicity island (cag-PAI) (11-16). According to the presence or absence of cagA, H. pylori strains are classified into two major sub-types, cagA-positive or cagA-negative (16, 17). Interaction between specific receptors on the gastric epithelial cells and the " needle "-like structure of Abstract Aim: Since, contradictory data have been reported about the effect of diverse variants of H. pylori virulence factors on IL-8 induction, we aimed to analyze the effect of this diversity on levels of IL-8 secretion in AGS cell line. Background: Helicobacter pylori colonizes the human stomach and induces the activation of inflammatory cytokines, including interleukin (IL)-8, in the gastric mucosa. This induction promotes neutrophil and monocyte recruitment that causes gastric tissue damage. Methods: To determine whether different strains of H. pylori and their CagA variants have possible roles on IL-8 induction, polarized AGS cell line was infected with CagA + H. pylori strains carrying different EPIYA motifs (ABCCC and ABC) and CagA-strain for 24 hours. Difference in stimulation of IL-8 was measured by ELISA. Results: IL-8 secretion was elevated in the treated cells with CagA encoding strains compared with the negative one. Furthermore, a noticeably increased level of IL-8 induction was measured by the CagA-EPIYA type ABCCC encoding strain in compare to that carried EPIYA type ABC Conclusion: Results of this study provide new evidence about different effects of H. pylori strains and possible roles of their CagA variants on IL-8 induction. It …


Introduction
Helicobacter pylori is colonized in the stomach of 35%-70% people worldwide. It is a Gram-negative bacterium that lead to disorders ranging from gastric inflammation to gastric adenocarcinoma (1)(2)(3)(4). Several virulence factors of H. pylori are involved in the pathogenesis of the stomach. H. pylori chronic infection could increase expression of specific immune mediators, such as interleukins, tumor necrosis factor (alpha), and interferons in the host cells (5)(6)(7)(8). Interleukin 8 (IL-8), a potent neutrophil chemotactic factor, plays a crucial role in the proliferation and migration of cancer cells in the infected patients. It also is able to stimulate the host specific inflammatory response through chemo attraction of neutrophils to the site of infected gastric mucosa and their activation (9,10).
In vivo and in vitro studies showed higher IL-8 secretion in the H. pylori infected cells and mainly correlated this elevated IL-8 level with expression of functional CagA, a major virulence factor of H. pylori which is located at the end of the cag pathogenicity island (cag-PAI) (11)(12)(13)(14)(15)(16). According to the presence or absence of cagA, H. pylori strains are classified into two major sub-types, cagA-positive or cagAnegative (16,17). Interaction between specific receptors on the gastric epithelial cells and the "needle"-like structure of type IV secretion system, T4SS, CagA is transferred into the cytosol, where it is phosphorylated by host cellular kinases on the repeat sequences known as EPIYA motifs, located in the carboxyl terminus of the protein (18)(19)(20). These process lead to an activation of signaling cascades inducing proinflammatory cytokines like IL-8 (13,(21)(22)(23). EPIYA motifs have been classified into four types, based on the adjacent amino acid sequences. The A and B motifs are found in all strains, while the C and D motifsare present within 'western' strains and East Asia, respectively (24,25). It has been suggested that East Asian CagA with one D motif is highly active and it has no requirement to increase its virulence, whereas the less active western type needs to increase its number of tyrosine phosphorylation motifs for being more virulent (26). Besides, the level of CagA phosphorylation depends on the number of EPIYA-C motifs (25,(27)(28)(29)(30). It has been shown that co-culture of AGS cell line with Asian strains increases IL-8 secretion on AGS cell line and it may induce more inflammation within the stomach which lead to an increase in atrophic gastritis and gastric cancer (26). Since, there are contradictory reports about the effect of H. pylori infection and the number of EPIYA motifs on interleukin (IL)-8 secretion, we aimed to determine the effect of CagA negative and CagA encoding H. pylori strains and its variants on IL-8 induction in an in vitro cell culture model.

AGS gastric epithelial cell co-culture and IL-8 ELISA:.
The human gastric cancer AGS (ATCC CRL-1739TM) cell line (IBRC, Tehran, Iran) were seeded into six-well plates at a density of 3×10 5 cells per well and incubated in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA), 1% nonessential amino acid (Gibco, Grand Island, NY, USA), 100 U ml −1 of penicillin and 100 μg ml −1 of streptomycin (Gibco, Grand Island, NY, USA) at 37•C in a humidified incubator (Memmert, Dusseldorf, Germany) containing 5% CO 2 for 2 days before the addition of H. pylori strains. For comparison of the effects of CagA positives and negative H. pylori strains, gastric epithelial cell lines AGS were infected with a CagA negative H. pylori strain and CagA positives strains at MOI 100. Briefly, two hours prior to infection, cells were washed with phosphate buffered saline (PBS, 1x) and the medium was replaced with fresh, antibiotic free DMEM media. Bacterial suspensions (100 μl) were used to infect gastric epithelial cells in 2 ml total volume. After 24 h incubation, the medium was removed, centrifuged at 15000 g for 10 min and the amount of secreted IL-8 into the medium was determined using Human IL-8 Elisa Ready-set-Go! ® Kit (2nd Generation, eBioscience, Korea), following the manufacturer's protocols. All samples were measured in duplicate in at least two independent experiments.

IL-8 levels in AGS cell line
In order to quantify the effect of H. pylori CagA variants and also and the number of EPIYAC motifs site on IL-8 induction, we infected AGS cells with different strains of H. pylori. The tested strains were divided into two groups, according to the presence or absence of cagA, positive strains (complete cag PAI, CagA EPIYA type ABC and complete cag PAI, CagA EPIYA type ABCCC), and negative strain (cag PAI -, cagA−). After 24 h co-cultured, IL-8 levels were measured as described above. As a control, IL-8 concentration in the supernatants of uninfected AGS gastric epithelial cell lines was determined in the same experimental layout. As presented in Figure 1, the IL-8 secretion was elevated in H. pylori infected cells irrespective of the negative or positive strain for cagA gene. Furthermore, IL-8 amount was five folds higher in the infected cell line with cagA EPIYA type ABCCC in compare to the infected cells with the cagA-EPIYA type ABC encoding strain

Discussion
Our results showed that IL-8 secretion was higher in infected cells in compare to uninfected cells, irrespective of the presence or absence of cagA gene. Infection with H. pylori is associated with an increased risk of gastric disease. H.pylori strains may induce more inflammation within the stomach and this may lead to an increase in atrophic gastritis and gastric cancer. After H. pylori infection, a number of genes especially inflammation genes are over expressed in host cells. IL-8, a chemo attractant for neutrophils, is a part of cancer progression. This cytokine release angiogenic growth factors, activates macrophage and immune responses at the tumor site, causes migration and survival of endothelial cells, and potentiates the epithelial-mesenchymal transition (9, 32). Ling, et al. showed that H. pylori infection correlates with up-regulated IL-8 expression and its level was found to be associated with invasion, lymph node spreading and clinical stages. These observations indicate that high levels of IL-8 may be associated with a poor prognosis and may be indicative of more aggressive gastric cancer (12). Therefore, it is tempting to consider IL-8 as a prognostic and predictive cancer biomarker. The result of this study also shows that although the amount of IL-8 secreted in this study was not so high, the IL-8 secretion was higher in infected cells with cagA positive strains than uninfected or infected cells with a negative one. Since our results demonstrated that CagA-EPIYA motifs ABCCC increased IL-8 level in compare to EPIYA motifs ABC, it seems that phosphorylation of CagA at terminal EPIYA-C motifs contributes to IL-8 secretion in AGS cells. However, there are studies that reported contrary results (33)(34)(35). Several in vitro and in vivo studies reported that H. pylori induces IL-8 through cagA-dependent and -independent manners (30,(36)(37)(38). Although different efficiency of IL-8 secretion was observed between different cagA-type strains, cagA was not always the responsible factor for IL-8 secretion (36,37).
In addition to cagA, H. pylori peptidoglycans also leading to IL-8 induction through activating NF-kB pathway (13,18,39). Papadakos et al showed that following infection of gastric epithelial cells with H. pylori strains, a higher activation of NF-kB was observed in the presence on phosphorylated EPIYA-C motifs and in lower levels, in the absence of cagA expression and CagA phosphorylation or in the cases where EPIYA-C motifs are totally absent (33) Others proposed that CagA multimerization (CM) motif, the highly conserved amino acid sequence FPLKRHDKVDDLSK, is contributed in IL-8 activation (40,41). Recently it has been suggested that CagL through activation of MAPKs and NF-kB induces secretion of interleukin-8 (IL-8) independently of CagA translocation and peptidoglycan (42). Zhang et al showed that the levels of IL-8 induced by H. pylori strains is noticeably differed at an early phase, irrespective of cagAspecific sequences. They suggested that other H. pylori factors, in addition to cagA, affect the enhancement of IL-8 secretion in early H. pylori infection phase (24). However, diversity of the level of secreted IL-8 by H. pylori remains a controversial issue, as a result of contradictory reports (43)(44)(45). More studies is needed in order to prove this argument.
In conclusion, we have demonstrated that the presence of functional CagA protein plays a central role in IL-8 secretion in epithelial cells and the level of IL-8 secreted is amplified with increasing numbers of CagA-EPIYAC motifs.